Figure 5.

ANKHD1 regulates proliferation via miR-29a-3p/miR-205 and to a lesser degree miR-196a. A, RCC4 cells were transfected with either nontarget (NT) or ANKHD1 siRNA (ANKHD1 si) and co-transfected with an miR-29a-3p inhibitor. The number of RCC4 cells undergoing mitosis was studied by flow cytometry measuring number of pH3-positive cells. B, experiments were repeated three times, and a p value of less than 0.05 was considered as significant using a one-way ANOVA. C, ANKHD1 was also silenced in 786-O cells using two independent siRNAs (top panels), and the number of mitoses was measured. Inhibition of miR-29a (left panels), miR-196a (middle panels), or miR-205 (right panels) was studied. D, to study how ANKHD1 control proliferation of 786-O cells, ANKHD1 was silenced using two independent siRNA, and the expression of CCDN1, VEGFA, and KLF4 was measured by qPCR. In addition to test whether the modulation of the target mRNAs is via miR-29a, the cells were co-transfected with a miR-29a-3p inhibitor. The results are normalized against HPRT expression and are shown as fold change when compared with nontarget siRNA-transfected cells. The experiments were repeated three times, and a p value of less than 0.05 was considered as significant using a one-way ANOVA. NS, not significant. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.